The long-term objective of this proposal is to determine how the chromatin response to a mammalian DSB, mediated by the phosphorylation of histone H2AX, regulates DSB repair. We discovered a critical role for H2AX in controlling sister chromatid recombination (SCR), a homologous recombination (HR) process that operates during S and G2 phases of the cell cycle to repair replication-associated DNA damage in an error free manner. In recent work, we discovered that MDC1, an H2AX-binding adaptor protein, is the major mediator of H2AX-dependent HR/SCR. We discovered that the chromatin response to a mammalian DSB encodes additional DSB repair functions, regulating both single strand annealing (SSA) and non-homologous end joining (NHEJ). Our work revealed a previously unsuspected degree of specificity to chromatin-based DSB repair functions and established the existence of a histone code for DSB repair. In this proposal, we will determine the biochemical basis of H2AX/MDC1-mediated HR/SCR, by analyzing a new MDC1-interacting protein complex involving TRIP12 and nucleophosmin (NPM1), which we have recently identified. We will also analyze other DSB repair functions encoded within the DSB chromatin response. Our Specific Aims are: 1. Define DSB repair functions of TRIP12 and NPM1. We will use genetic, biochemical and cell biological techniques to accomplish this Aim. 2. Define roles of H2AX and associated proteins in SSA and in NHEJ. We have developed novel mammalian reporters for SSA and NHEJ that deliver rapid, specific and quantitative flow cytometric measurement of these DSB repair outcomes. We will use these reporters to determine the contribution of H2AX and its associated proteins to SSA and NHEJ. 3. Determine the mechanisms by which 53BP1 mediates NHEJ. We will use NHEJ reporters that we developed previously, as well as new reporters described herein, to determine how 53BP1 executes NHEJ. We will combine this with use of a novel multi-photon laser for DSB induction and real time imaging, as well as conventional DSB inducing agents in 53BP1-/- cells to study how 53BP1 regulates NHEJ. Success in the projects described in this proposal will: 1. Elucidate a chromatin-based pathway for regulating HR/SCR. 2. Identify TRIP12 and NPM1 as novel chromatin-associated components of mammalian HR. 3. Validate new, rapid and specific reporters of single strand annealing (SSA) and NHEJ. 4. Reveal how 53BP1 regulates NHEJ 5. Validate a novel multi-photon laser and real time imaging tool to study the kinetics of DSB repair.